Press room ... 1997 archive

Highlights from the Clinical
Immunology Society HIV Symposium

March 3, 1997

San Francisco, CA—On Sunday, 23 February 1997, the Clinical Immunology Society hosted a day long HIV symposium during the joint meeting of the Clinical Immunology Society, the American Academy of Allergy, Asthma and Immunology and the American Association of Immunologists (CIS/AAAAI/AAI). Discussion ranged from a broad overview of work on immune pathogenesis and immune-based therapies, to detailed discussions of the newly identified co-receptors (CKR5 and CXCR4, also known as fusin or LESTR) for HIV, and the beta chemokines which ‘bind’ to them (RANTES, MIP1-alpha, MIP1-beta and SDF-1). Project Inform has covered these topics in the past (see PI Perspective 19, Opening the Door on HIV, PI Perspective 20, Immune-Based Therapies, and the Report from the Institute of Human Virology, What’s New?) This report highlights the following new information presented at the symposium:

The state of the thymus in HIV disease,
CD8+ cell dysfunction,
immune reconstitution as a product of triple-drug therapy
and thalidomide proves effective in treating HIV-related mouth ulcers.
The State of the Thymus in HIV Disease
Dr. J. Michael McCune of the Gladstone Institute in San Francisco has been studying the state of the thymus in HIV disease. The thymus is an important organ critical for T cell maturation. All cells originate from the bone marrow, yet where a cell matures determines its function. Cells which mature in the bone marrow are called B cells (B = bone marrow), while cells that mature in the thymus are called T cells (T = thymus). Both CD4+ and CD8+ T cells mature in the thymus.

When someone has increases in CD4+ or CD8+ cell counts, there are a few places these cells could be coming from. They may come from redistribution of existing cells, new development through the thymus or from the expansion of existing cells.

Redistribution - The majority of CD4+ and CD8+ cells reside in the lymph nodes. At any time, only about 10% of cells are circulating through the blood in the periphery, which is where they are collected and measured from when someone has laboratory work done. If even a small percentage of cells are ‘released’ from the lymph nodes, this could look like a dramatic increase in cell numbers, when actually they are only being redistributed.
Thymus pathway - Another place new cells can come from is through the thymic pathway. Bone marrow cells mobilize and begin dividing. Some of the daughter cells of the bone marrow cells migrate to the thymus where they are educated to become T cells. T cells, which mature through the thymus, unlike those which undergo reproduction in the blood, are capable of recognizing new infections. Therefore, the thymus is a critical organ for reconstituting immune responses lost due to HIV infection and educating new cells. It may take six months to a year for any significant number of bone marrow cells to mobilize and begin maturing through thymus. Therefore, any rapid increase in CD4+ or CD8+ numbers in adults is most likely due to either redistribution or reproduction of existing cells.
Expansion of existing cells - Cell increases may also come from existing cells which divide and reproduce. This is probably where the majority of new cells come from in adults. Dr. Cliff Lane has likened CD4+ cells to a box of scrabble tiles. Each tile looks the same, but when you turn them over they each have a specific function. If all the P’s in the scrabble tile set have been destroyed by HIV infection, then there are no P’s to reproduce and the ability to fight PCP may be permanently gone without new cells coming through the thymus.
The thymus is critical for immune reconstitution. Research on the state of the thymus in people with HIV underscores the challenges faced in immune restoration. In children the thymus is a very sizable organ, covering a large area beneath the breast plate, about the size of a bib. As we age, the thymus shrinks and in adults it is about the size of a small fist. While a number of studies suggest that the thymus becomes less active as people age, other studies have shown that though much smaller, the thymus remains functional. Studies in the pediatric HIV setting suggest that the thymus is infected by HIV. Children with HIV show evidence of thymic dysfunction and these children tend to progress more rapidly in HIV-disease. Because the thymus lays behind the rib cage, it is very invasive to biopsy to assess the state of the thymus and whether or not the thymus is infected with HIV.

Dr. McCune, therefore, conducted a number of CT scans (similar to an X-ray) to look for the presence of thymus in adults with HIV infection. Dr. McCune conducted CT scans on nearly 100 people in varying age ranges (20–29, 30–39, 40–49) and at various CD4+ cell counts (above and below 420 CD4+ cell counts). These scans showed a correlation between higher CD4+ cell counts, younger age and the presence of thymus material. In every age group, people with greater than 420 CD4+ cells were significantly more likely to have evidence of thymus mass on the scans. There was also an association between older age and decreased presence of thymus material. Not surprisingly, there was an association between the fraction of CD4+ and CD8+ cells which were naïve (i.e. showing evidence of having matured through the thymus pathway and capable of mounting new immune responses), total CD4+ and CD8+ cell counts, the presence of thymus material and stage of HIV disease. In other words, those with evidence of thymic mass tended to be younger, have higher CD4+ and CD8+ cell counts, have a larger percentage of naïve cells and were generally healthier. Based on these findings, Dr. McCune speculates that in people with intact thymus material, immune restoration may be possible with aggressive triple-drug antiviral therapy, including a protease inhibitor and/or a non-nucleoside reverse transcriptase inhibitor (NNRTI). In those without evidence of thymus material, other strategies, such as thymus transplantation, may need to be employed. What he does not yet know, however, is whether the thymus regenerates after viral replication is controlled and CD4+ cell counts rise. These studies, looking at the effect of antivirals on the thymus, are just now being conducted.

What was perhaps most surprising from Dr. McCune’s work was that nearly 50% of people had thymus material. This is a much greater percent than expected even among healthy HIV-negative adults. Dr. McCune will be expanding the study and evaluating thymus presence in HIV-negative adults across various age ranges.

CD8+ Cell Dysfunction
While no one disputes the importance of CD8+ cells in controlling HIV infection, there is no clear consensus on how to measure their function. Dr. Janis Gorgi of the University of California at Los Angeles has presented information which shows that increases in activated CD8+ cells (CD8+CD38+) correlate with increased HIV disease progression. Work by Dr. Judy Lieberman suggests that while there is a large increase in the number of CD8+ cells after someone becomes infected with HIV, many of these cells have defects in cell surface proteins which may impair their function. Moreover, no one understands why CD8+ cells become depleted in people with advanced HIV disease, as presumably CD8+ cells do not become infected by HIV. Recent reports at the meeting of the Institute of Human Virology in Baltimore suggest that some CD8+ cells may become infected with HIV and die very rapidly.

Dorothy Lewis, of Baylor University, presented information on another potential mechanism for CD8+ cell dysfunction in HIV disease. Dr. Lewis first presented on the importance of CD8+ T cells in HIV disease. CD8+ cells are undoubtedly critical in control of viral replication after someone is first infected with HIV. CD8+ cells which inhibit HIV replication are called cytotoxic lymphocytes, or CTLs. A number of groups have shown that CTL function appears to be intact and strong in people with HIV who are long-term non-progressors, and therefore may play an important role in controlling HIV disease progression. While there is a profound increase in CTL activity very early on in the disease process, there is a profound reduction in this activity as people progress. Some people have suggested that CD8+ cell numbers may be more important than CD4+ counts. CD8+ cells do not thrive or function properly without the support of CD4+ cells, however. This has been well documented by Dr. Jay Levy and others who do research on CD8+ cells. Dr. Levy has presented work showing that CD8+ cells produce a factor which inhibits HIV replication, yet the number of CD8+ cells required to inhibit this replication increases dramatically as CD4+ cell numbers decline. Moreover, people doing studies with CD8+ cell therapy find that unless they infuse people with factors which are produced by CD4+ cells, CD8+ cells often die within days in people with HIV. CD8+ cells are clearly important in controlling HIV disease, and they need the support of CD4+ cells in order to do their job.

When looking at CD8+ cells from HIV+ people across various stages of disease, there is evidence of dysfunction in most people. As people progress in HIV disease, there appears to be a loss of naïve CD8+ cells (cells capable of recognizing new infections) that parallels a loss in naïve CD4+ cells, despite the rise in total number of CD8+ cells. There is an increase in activation markers which correlate to HIV disease progression (CD8+CD38+, CD8+DR+). CD8+ cells also appear to lose an important cell surface protein called CD28. When the cells are studied in test tubes, they are sluggish and do not function or react well (they become anergic). Also, in test tube studies, these cells appear to be programmed to self destruct, or apoptose.

Dr. Lewis focused on illuminating the importance of the loss of CD8+ cells bearing the CD28 protein (CD8+CD28+). She noted a correlation between the loss of CD4+ cells and a decrease in percentages of CD8+CD28+ cells. A decline in CD8+CD28+ cells did not correlate with total CD8+ cell numbers and this loss in CD8+CD28+ cells correlated with disease progression and increased HIV replication.

CD28 is an important cell surface protein for controlling immune activation, which is associated with HIV disease progression. It is also important in stabilizing the production of interleukin-2 (IL-2) an important chemical, produced by CD4+ cells, which helps CD8+ cells to thrive. As the percentage of CD8+CD28+ cells decline, CD8+ cells appear to become anergic and self destruct. Those CD8+ cells which lack CD28+ have reduced ability to reproduce and impaired development.

The key question, of course, is what causes CD8+ cells to lose the CD28+ protein. Dr. Lewis suggests that CD28+ expression is altered by immune chemicals, called cytokines. Interleukin-4 (IL-4), is a cytokine which is important in supporting antibody responses. Some studies suggest that IL-4 production increases as HIV disease progresses. IL-4 downregulates CD28+ expression while increasing CD8+ cell number. This is consistent with what happens in HIV disease, as CD8+ cell numbers rise dramatically after infection. Further, CD28 is downregulated by interactions between CD4+ cells and other immune cells, called antigen presenting cells. In test tubes, Dr. Lewis found that by adding IL-2, a factor produced by CD4+ cells, it is possible to restore CD8+CD28+ cell function. This is consistent with what is seen in HIV disease, as IL-2 levels decrease, there is an apparent decrease in HIV-specific CTL activity and a loss of control of HIV replication by the immune system. The clinical importance of this would suggest that as studies of IL-2 in HIV disease move forward, researchers should be examining the impact of therapy on CD8+CD28+ cells.

Immune Reconstitution as a Product of Triple-drug Therapy
Recently Project Inform reported on a number of small studies which looked at the immunologic consequences of aggressive antiviral therapy (see What’s New?, Report for the 4th Conference on Human Retroviruses and Opportunistic Infections). Dr. Michael Lederman of Case Western University in Cleveland, Ohio presented preliminary data on the largest study of this kind at this CIS/AAAAI/AAI meeting. People receiving triple-drug therapy, including a protease inhibitor or an NNRTI, have experienced dramatic reductions in HIV RNA levels as well as increases in CD4+ cell counts. Despite these increases in CD4+ cell numbers, there have been reports of people developing cytomegalovirus disease (CMV disease) at abnormally high CD4+ counts after initiating triple drug therapy, making some question the function of these new cells. While these reports have been discouraging, there have been an equal if not greater number of reports of people clearing cryptosporidium which was unresponsive to therapy, clearance of Kaposi’s Sarcoma lesions, clearance of PML and other opportunistic infections and dramatic improvements in overall general health. These data would suggest that the new cells are functioning. The AIDS Clinical Trials Group (ACTG) developed a study to rigorously examine the immunologic changes that occur after initiating highly active antiretroviral therapy (HAART).

The ACTG study, ACTG 315, is examining viral and immune parameters of 53 people with CD4+ cell counts ranging from 100 to 300. All study participants were tolerant to 3TC prior to entering the study. Everyone stopped all antivirals for one and a half months prior to entering the study. Volunteers received ritonavir alone, in a dose escalating fashion, for 10 days, and then added AZT and 3TC. At the time of this presentation, data were available on about 34 of the participants.

The following measurements were looked at:

Delayed type hypersensitivity responses - a simple way to examine cell function.

Lymphoproliferative assays - this looks at the cell’s ability to reproduce in the presence of stimulation. If cells do not reproduce when stimulated by common infections that they should recognize, this suggests that they are not functioning properly.

NK activity - natural killer cells destroy cells, randomly, which may be infected with HIV and other pathogens.

Flow cytometry - flow cytometry is used to measure CD4+ and CD8+ cells. It can also be used to look at other cells, such as naïve T cells, capable of mounting new immune responses, and memory T cells, which are already committed to recognize various infections. There are naïve cells in the peripheral blood, which can reproduce. These naïve cells are different from naïve cells which mature through the thymus, as new thymus naïve cells are not yet ‘educated’. Naïve cells expanding in the periphery are capable of mounting responses, but they are likely already educated to recognize particular infections. Only naïve cells maturing through the thymus are capable of mounting truly new immune responses and having diverse T cell receptors (e.g. new configurations of V-beta).

V-beta analysis of T cell receptors - this analysis looks at T cell repertoire. Using flow cytometry, researchers look at the diversity in the variable region of the beta chain of the T cell receptor on CD4+ and CD8+ T cells. This analysis tells us something about the diversity of T cells. Healthy HIV-negative individuals have a wide range of V-beta configurations. People with HIV seem to lose this range of configurations early on, in both CD4+ and CD8+ cell populations. When there are increases in CD4+ cells, but no increase in the diversity of this repertoire, it is believed that either existing T cells are expanding, or new T cells capable of new functions are being destroyed before they leave the thymus.
The average viral levels (HIV RNA) at the start of the study was 87,000. By week 12, 3 months after initiation of therapy, the average viral level was 150, representing a 2.85 log reduction in HIV RNA. By day 84 (almost 3 months) CD4+ cell counts rose from an average of 164 to 309, and CD8+ cell counts rose from 685 to 1000.

Delayed type hypersensitivity and skin test responses - Of the 36 volunteers who could be evaluated at 12 weeks, 3 had delayed type hypersensitivity responses prior to initiating therapy. Of these 3, 2 people lost this reactivity by week 12 and no one who did not have a response prior to therapy mounted one by week 12. Researchers also looked at skin test reactivity, using a panel of common skin test reactants, such as tetanus and candida. A skin test is when researchers put small quantities of material (called antigens), such as tetanus, under the skin. If the immune system recognizes these antigens then an inflammatory response is induced and the area turns slightly swollen and red. This signifies that immune functions are intact. If people don’t have this response, they are said to be anergic, which means that their cells have diminished ability to interact and respond. Prior to initiation of therapy, 33 of 36 volunteers were anergic. By week 12, 10 of the 36 had developed new or increased responses. This skin test research would suggest that there is an improvement in immune function after initiating HAART.

Lymphoproliferative assays - When researchers looked at the cell’s ability to respond to stimulation, interestingly they saw no increases in responses to a number of stimuli, including stimulation with HIV proteins, at week 4 and 12 after initiation of therapy. About 50% of the people, however, did see increases in responses to a few specific stimuli, including candida, which is the agent which causes fungal infections. This would suggest that some, but not all, immune responses are restored or enhanced when CD4+ cells increase as a product of HAART.

Flow cytometry - By looking at various cell populations using flow cytometry, researchers noted a significant increase in CD4+, CD8+ and B cells, and no change in the number of natural killer cells at day 84 after initiation of therapy. Table 1 outlines the breakdown in the increase in CD4+ and CD8+ naïve and memory cells:

Table 1
Day CD4+ CD4+ naïve CD4+ memory CD8+ CD8+ naïve CD8+ memory
0 164 27 80 685 112 287
84 309 54 165 1000 253 343
Is this change significant? yes yes yes yes yes no

While the increase in CD4+ naïve cells was significant, 27 to 54, a doubling, the numbers are still very low. This corresponds to data recently reported by a French group which showed naïve cells remaining relatively low and only beginning to rise after 12 months of aggressive therapy. The majority of new cells were memory cells, which means that their function is already determined and are unlikely to be able to make new responses.

Investigators also examined the effect of HAART on markers of immune activation, which is associated with HIV disease progression. Prior to the initiation of HAART, 26% of CD4+ cells and 60% of CD8+ cells expressed markers suggesting the cells were activated. After about 3 months of HAART, those numbers dropped to 13 and 31 percent respectively, signifying a decrease in immune activation of about 50% after 3 months. Similarly, when looking at Tumor Necrosis Factor - alpha (TNF-alpha) in the plasma, levels fell from 58 pg/ml to 35 pg/ml after 3 months of therapy. TNF-alpha is also associated with immune activation, HIV replication, KS disease progression, AIDS related wasting and HIV disease progression. This decrease in immune activation suggests that HAART improves immune status.

V-beta analysis - When looking at V-beta repertoires, researchers studied both HIV-negative as well as HIV-positive individuals. Healthy HIV-negative people have a broad distribution of V-beta families in their T cell receptors. People with HIV show quite a perturbed repertoire by comparison. In examining the effect of HAART on V-beta diversity, the study showed that the families remained fairly constant over time in both the healthy and the HIV-positive people on therapy. While there was an increase in numbers within each family among the HIV+ people receiving HAART, there was not a broadening of the diversity of the T cell receptor as a response to therapy, after about 3 months.

These data, taken together with the work presented by Dr. McCune on the state of the thymus in HIV disease support the following conclusions:

• People should get tested for HIV and learn about HIV and available therapies. There are clearly therapies out there which can profoundly impact the disease process. The option to intervene with these therapies early, perhaps when they can make the most impact, depends on someone finding out their HIV status while their immune system is still intact.
• HAART should be begun early, before serious immune damage has taken place and while the thymus is still intact.
• People with T cell increases as a consequence of HAART should maintain preventative therapy against opportunistic infections. While there are seeming improvements in immune status and some tests suggest improvements in immune function, these tests are somewhat contradictory and thus OI prophylaxis should be maintained.
• Finally, treatment strategies to enhance immune response, in conjunction with HAART, must be explored.

The key questions which the ACTG study aimed to answer were:

• How does HAART increase CD4+ cells? Are the new cells functional? Currently we do not know the answer to this question. It is unclear if the new cells are newly produced through the thymus or if they are existing cells which are reproducing. If they are new cells, however, one would expect to see a broadening of the V-beta families in the T cell receptor. However, it could be that cells with new V-beta families are being destroyed in the thymus.

• Does HAART increase cell populations other than CD4+ cells? HAART appears to significantly increase CD4+, CD8+ and B cells, but has no effect on natural killer cells after 3 months. Increases are seen in both CD4+ and CD8+ naïve cells after 3 months, but increases in memory cells seem restricted to the CD4+ population.
• Does HAART decrease the immune activation association with HIV disease progression? After 3 months, HAART appears to decrease immune activation associated with HIV disease progression. This is consistent with a French groups findings, who saw activation markers continue to decrease well after 4 months of HAART.
• Does HAART result in immune restoration? HAART appears to partially result in immune restoration, as clearly there is some evidence of improvements in immune function. A French group showed that after 12 months of HAART, naïve cells began to increase more noticeably. It may be that after a longer period of time, new cells will mobilize through the bone marrow and into the thymus pathway. As noted earlier, in bone marrow transplant patients this kind of immune restoration can take from 6 months to a year. It is unknown at this time if more substantial immune restoration will take place after a longer period of continued HIV suppression.

Thalidomide Proves Effective for
Treating HIV-related Oral Aphthous Ulcers (mouth ulcers)
Thalidomide was studied in AIDS Clinical Trials Group study 251 for its effect on oral and esophageal ulcers. The part of the study examining the effect of treatment on esophageal ulcers is still ongoing. The study enrolled 45 volunteers who received 200mg thalidomide or placebo daily for 4 weeks. Those whose mouth ulcers completely disappeared were given a maintenance dose of thalidomide of 100mg three times a week, or placebo for an additional 24 weeks. Those who did not achieve complete clearance of mouth ulcers, be they originally assigned to placebo or thalidomide, were offered an additional 4 weeks of thalidomide at 200mg daily. If mouth ulcers continued to persist after this second 4 week phase of the study, participants were then offered 400mg thalidomide daily for 4 weeks. All participants entered the study with biopsy confirmed ulcers which had persisted for 2 weeks, which were greater than 5mm in diameter. Ulcers had to be confirmed negative for herpes simplex virus and participants had to be on stable antiretroviral therapy. Because of the birth defects associated with thalidomide, women participating in the study either had to be sexually abstinent or use 3 forms of birth control, including a hormonal method and 2 barrier methods. The median CD4+ cell count was 30 in the placebo group and 22 in the thalidomide treated group.

Table 2 summarizes the results from this study
Placebo Thalidomide
Number of participants 22 23
Complete response to therapy after 4 weeks 1 14 (61%)
Complete OR partial response after 4 weeks 4 21 (91%)

The median time to ulcer healing was 3 weeks. Major side effects of thalidomide include birth defects if used during first trimester pregnancy, fatigue/drowsiness and rash. Peripheral neuropathy, characterized by pain and tingling in the hands and feet, is also a side effect of thalidomide, yet in this study it was not seen more in the thalidomide group compared to those receiving placebo.

Thalidomide has been shown, in other studies, to decrease TNF-alpha levels, which are associated with increased HIV replication, Kaposi’s Sarcoma progression, wasting syndrome and HIV disease progression. Interestingly, in this study, those receiving thalidomide saw an increase in TNF levels, as well as an increase in viral levels (HIV RNA) of .4 logs over their pre-study levels. Those receiving the placebo saw slight reductions in TNF levels and only a .05 log increase in HIV RNA levels by week 4. In the population studied, thalidomide failed to inhibit TNF and actually enhanced HIV replication. These data suggest that any long-term use of thalidomide in HIV should be approached with caution.

In closing, Dr. Peggy Johnson gave an impassioned plea for immunologists to get involved in vaccine research. With the recent advances in antiviral therapy, there is a wave of optimism in the United States and other developed nations where many people have realized dramatic improvements in their health status as a consequence of aggressive triple-drug therapy including a protease inhibitor or an NNRTI. Sadly these advances will probably never reach many developing nations, where the epidemic continues to rage, unabated. In nations where the per person health care budget is literally a few dollars, combination therapy approaches which cost thousands of dollars a year simply will not be accessible. People in most developing nations still do not have access to AZT, much less these more potent antivirals.

At some STD clinics in sub-Saharan Africa, 75% of clients are HIV-infected. In some clinics delivering prenatal care to pregnant women in large cities in Africa, the HIV prevalence is 35%. It is estimated that nearly 1 of every three people in sub-Saharan Africa is HIV+. In Africa, women seem to be much harder hit by the epidemic then men, for every 2 men who are HIV+ there are 3 HIV+ women. The World Health Organization estimates that over 30 million people have been infected with HIV world-wide. Approximately 25.5 million of these people are still alive, and approximately 6 million are living with more advanced stages of AIDS. These numbers are already staggering and yet continue to rise daily, most profoundly in southeast Asia. It is estimated that globally there are 7,500 infections each day, which means that every hour more than 300 people are becoming infected. By the year 2000, it is estimated that AIDS will be the third leading cause of death world-wide. While in the United States we ponder the possibilities of improving on the gains realized with triple-drug therapy, for the majority of people living with HIV these ponderings are a luxury that will never be afforded.